This invention relates to immobilized glucose isomerase, and particularly relates to the treatment of a flocculated homogenate of whole cell glucose isomerase, derived from microorganisms of the Actinoplanes genus, with a combination of milled granular carbon of about 50-100 mesh size and a smectite filler added prior to drying.
This application involves an improvement of the process and product described in Ehrenthal, Shieh, Scallet and Rajpara, U.S. Pat. No. 4,242,451 issued Dec. 30, 1980, entitled METHOD OF TREATMENT OF FLOCCULATED HOMOGENATE OF MICROBIAL CELLS CONTAINING GLUCOSE ISOMERASE.
U.S. Pat. No. 4,242,451 (assigned to the assignee of this invention) discloses a process for treating a flocculated homogenate of whole cell glucose isomerase derived from microorganisms of the Actinoplanes genus in order to dry the homogenate so that the enzyme is immobilized and usable in converting glucose to fructose. The critical step in the process of U.S. Pat. No. 4,242,451 is the addition of a water absorbing smectite filler to the flocculated homogenate prior to drying and/or extrusion. The system of U.S. Pat. No. 4,242,451 basically mixes a concentrated flocculated homogenate with prescribed amounts of bentonite, extrudes this material, dries it, mills it and screens the immobilized glucose isomerase to the appropriate mesh size. The immobilized glucose isomerase from U.S. Pat. No. 4,242,451 has a half life of 50-60 days at 60.degree. C. and a productivity of 5000-6000 lbs. dry weight high levulose syrup (HLS) per lb. of immobilized glucose isomerase as is, for the first half life of the enzyme.
One of the principal objects of the present invention is to significantly reduce the cost of immobilized glucose isomerase compared to that made by the process of U.S. Pat. No. 4,242,451. Another object is to improve the properties of immobilized glucose isomerase as compared to the properties of the immobilized glucose isomerase made by the process of U.S. Pat. No. 4,242,451.
As noted in the specification of U.S. Pat. No. 4,242,451 there are several prior patents which discuss the flocculation of glucose isomerase cells with various flocculation agents. These processes, as noted in U.S. Pat. No. 4,242,451, are not suitable for use with microorganisms of the Actinoplanes genus, because the Actinoplanes cells are difficult to dry and are difficult to extrude since they retain a large amount of water. The retained water cannot be removed effectively by conventional drying means.
Among the patents noted as prior art in U.S. Pat. No. 4,242,451 are:
Aotz et al, U.S. Pat. No. 3,980,521 which discloses a method of preparing a water insoluble glucose isomerase product by concentrating and homogenizing microorganism cells to form a homogenized cell concentrate containing ruptured cells, reacting the homogenized concentrate with glutaraldehyde to form a coherent solid product, and removing water and shaping the coherent product into a divided form.
Lee et al, U.S. Pat. No. 3,821,086 discloses a process for converting glucose to fructose by contacting a glucose substrate with flocculated whole microbial cells containing glucose isomerase. The whole microbial cells are previously flocculated by means of a polyelectrolyte flocculating agent.
Long, U.S. Pat. No. 3,989,596 discloses a method of forming an enzyme-containing aggregate comprising the steps of flocculating microbial cells and their associated enzymes with a synthetic polyelectrolyte flocculating agent, and drying the aggregate.
Lee, U.S. Pat. No. 3,989,597 discloses a method of forming an enzyme-containing aggregate comprising the steps of flocculating microbial cells and their associated enzymes with a synthetic polyelectrolyte flocculating agent, freezing the aggregate, then thawing and crushing the aggregate.
Lee, U.S. Pat. No. 3,821,086, Long, U.S. Pat. No. 3,989,596, and Lee, U.S. Pat. No. 3,989,597 also state that the flocculated cells may be extruded into various shapes suitable for use in an enzymatic process.
In U.S. Pat. No. 4,242,451, water retention of organism of the Actinoplanes genus was attacked by the addition of a smectite filler to the flocculated homogenate prior to drying and/or extrusion.
In the present process we have unexpectedly discovered that adding 50-100 mesh milled granular carbon to the flocculated homogenate increases the productivity of the immobilized glucose isomerase by about 100% compared to the product of U.S. Pat. No. 4,242,451.
The recently issued U.S. Pat. No. 4,289,853 to Barley et al is entitled "HIGH LOADING OF IMMOBILIZED ENZYMES ON ACTIVATED CARBON SUPPORTS" and relates to the treatment of activated carbon with carbodiimide or an isoxazolium salt to form active organic radicals on the surface of the carbon followed by treatment of the reacted carbon with an enzyme to immobilize the enzyme on the carbon support by displacing the salt or the carbodiimide and resulting in a carbon-enzyme product. U.S. Pat. No. 4,289,853 also discusses the state of the art as to using carbon in enzyme immobilizing techniques.
It should be noted that U.S. Pat. No. 4,242,451 suggests adding carbon to the smectite filler to increase porosity of the extruded product (col. 2, lines 52-54). Also Tables III and IV of U.S. Pat. No. 4,242,451 show addition of carbon (Darco G-60 from ICI-USA). This carbon is stated to increase available activity as compared to filter aid (Celite 560 from Johns-Manville). However, Darco G-60 is powdered carbon with 70% passing through 325 mesh screen. Conventional granular carbon is accepted to be 12-30 or 40 mesh.
We have found that when the granular carbon is milled to 50-100 mesh and added in an amount of about 5% to about 55% by weight based on the weight of the dry solids of the total mass prior to extrusion and/or drying which includes the smectite, we obtain an unexpected improvement in productivity and stability (half-life) of the immobilized glucose isomerase.